The objective of this project is to understand the genetic basis of speciation at the molecular level by analyzing the sterility in hybrid males. The central questions is whether there exist major genes that cause complete male sterility in the interspecific hybrids of Drosophila and, if they do, how these genes can be characterized at the molecular level. We proposed to introgress small segments of the X chromosome from D. mauritiana or D. sechellia into the genome of D. simulans by repeated backcrossing. The introgressions will be identified fist by a subset of nine visible markers and then by a series of X-linked DNA clones. 1. Fine mapping and molecular cloning of the Ods gene-We have demonstrated that a major factor for hybrid male sterility, Ods, exists in 16DE, a 500kb interval within which we have 22 crossover points (Appendix 1). We propose to demarcate Ods within a letter division by using the PCR-SSCP analysis based on the primer sequences derived from cosmic clones. 10-20 more introgression lines with a crossover point in this finer interval will then be screened by performing PCR-SSCP at either of the two flanking DNA sites. Overlapping clones covering the whole letter interval will be isolated. The goal is to define Ods within 20kb. 2-3. Cytological, molecular and further genetic analysis of Ods-Light microscopy and 2- dimensional protein gel electrophoresis will be performed. When Ods is accurately mapped, transcript analysis and, eventually, germ line transformation will be carried out. Genetic interactions with neighboring genes, polymorphic suppressor in D. simulans and EMS-induced mutations will be analyzed. 4. A complete analysis of the sterility of the y-rb-np-region introgressed from both D. mauritiana and D. sechellia will be carried out. The analyses of this second region will be as extensive and detailed as those for the Ods genes, including genetic, cytological and molecular characterizations. 5. The sterility associated with the region marked by np-v-m-g will be delineated by coarse mapping at the resolution of letter divisions. When all the proposed experiments are completed, 80% of the X chromosome will have been analyzed. 6. We will analyses the effect of Y chromosome on hybrid sterility by introgressing each of 5 Y chromosomes from D. simulans into the two sibling species and, reciprocally, from D. mauritiana into D. simulans (Appendix 5,6). It will be the most comprehensive analysis of the role of Y chromosome in the sterility of hybrid males.